OA08.4. Spatial Transcriptomic Analysis of Non-Dysplastic Barrett's Esophagus From Patients Who Did and Did Not Progress to Advanced Disease

Transcriptomic Changes and Biomarkers in Barrett’s Metaplasia, Dysplasia and Cancer

P1.108. Molecular Determinants of Tumor Morphology in Esophageal Adenocarcinoma

Esophageal Cancer: Molecular Biology/Pathology Esophageal adenocarcinoma (EAC) is histologically graded as well, moderately, or poorly differentiated. Poorly differentiated tumours are associated with worse prognosis compared to well and moderately differentiated tumours. However, the molecular mechanisms driving these differences are poorly understood and their delineation may lead to improved biologically-informed patient stratification and therapeutic development. We primarily analyzed two cohorts of laser-capture microdissected (LCM) EAC samples: a discovery cohort consisting of 73 untreated primary biopsies and a validation cohort of 34 metastatic and/or post-treated samples. Additional validation cohorts included an independent cohort from The Cancer Genome Atlas (TCGA; n=80), a spatial transcriptomics (Visium) cohort (n=6), and a patient-derived EAC organoids cohort (n=24). Consensus non-negative matrix factorization (NMF) was performed on the discovery cohort (K=9 components, 3 glandular, 2 transitional, 1 signet ring associated, and 3 others) and independently on TCGA (K=9, 3 glandular, 1, transitional, and 5 other). The discovery NMF was projected onto the validation cohort. Morphology scores were calculated by summing sample component weights within their predefined component groups and correlated with histologic quantification by a board-certified gastrointestinal pathologist. The discovery NMF model was validated in TCGA, where all three glandular signatures and one transitional signature were reproducible. The second transitional signature was confounded by squamous contamination in TCGA samples. The signet ring-associated component was not detected in TCGA and was not observed in corresponding slide images. NMF-derived morphology scores accurately predicted tumour histology (Figures 1,2), with mean absolute errors (percentage points) of 16.05%/14.89% (glandular), 13.59%/15.36% (transitional), and 8.18%/9.41% (signet ring) in discovery/validation cohorts, respectively; these errors seem reasonable given potential LCM bias and the inherent limitations of pathologist quantification. Signatures were further validated by spatial transcriptomics with pathologist annotations, multi-area within-slide LCM RNA-seq, and patient-derived EAC organoids. Importantly, the transitional and signet ring signatures were associated with worse prognosis compared to tumors with a glandular signature. We report the first validated morpho-molecular classification of EAC. Our findings highlight the limitations of bulk RNA-seq, particularly the overinflation of poorly differentiated signatures due to the presence of squamous contamination and demonstrate the value of profiling following LCM. Ongoing studies are integrating whole-genome sequencing and clinical correlates to further define the biological and therapeutic implications of these morpho-molecular subtypes.

Spatial transcriptomics reveals molecular differences associated with malignant transformation in oral epithelial dysplasia

Background Oral epithelial dysplasia (OED) is a precancerous oral lesion with variable risk of progression to oral squamous cell carcinoma (OSCC). The molecular basis underlying this progression remains incompletely understood. To address this gap, this study applied spatial transcriptomics to characterize benign, OED and OSCC biopsies with a focus on the comparison between transforming and non-transforming OED. Methods Spatial transcriptomic profiling was performed on 13 benign, 15 OED (8 transforming, 7 non-transforming), and 14 OSCC biopsies using the NanoString GeoMx Digital Spatial Profiler. Regions of interest were segmented into epithelial and immune-enriched compartments with morphological markers. Gene expression was measured using the GeoMx Cancer Transcriptome Atlas (~1, 800 genes) and differentially expressed genes (DEGs) were identified using linear mixed-effects modeling. Exploratory bioinformatic analyses were performed to provide biological context. Results Comparison of OED with and without transformation identified a limited set of 11 epithelial DEGs, including genes associated with antigen presentation and interferon signaling (e.g., B2M, STAT1, and CD74), while no significant DEGs were detected in immune-enriched regions. Pathway analyses indicated enrichment of immune- and interferon-related processes. Given the modest sample size and targeted gene panel, these findings should be considered exploratory in nature. Conclusions This study provides spatially resolved, exploratory insights into molecular differences between OED lesions with distinct clinical outcomes. The results suggest altered epithelial-immune interactions in transforming lesions, though these findings require validation. Spatial transcriptomics may offer a useful framework for investigating early molecular changes in oral carcinogenesis.

Spatially resolved proteomics provides insights into the normal-dysplasia-tumor continuum in oral squamous cell carcinoma.

BACKGROUND Oral epithelial dysplasia is a recognized precursor lesion for oral squamous cell carcinoma (OSCC), yet the proteomic changes underlying the progression from normal epithelium to dysplasia and ultimately to OSCC remain incompletely characterized. This gap is partially due to the high interpatient heterogeneity of OSCC, the frequent reliance on cross-patient comparisons, and the omission of dysplasia as a critical intermediate stage. METHODS A histology-guided, spatially resolved proteomics workflow was applied to formalin-fixed paraffin-embedded tissue blocks from six patients with primary OSCC. For each case, spatially contiguous normal (N), dysplasia (D), and tumor (T) regions from the same lesion-bearing block, yielding 18 regional proteomes. Proteins were profiled using data-independent acquisition liquid chromatography-tandem mass spectrometry. Downstream analyses included differential expression analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment, soft-clustering trajectory analysis, protein-protein interaction analysis, exploratory clinicopathological stratification, and immunohistochemistry validation. RESULTS The spatially matched N-D-T dataset identified 1,683 proteins in N, 2,100 in D, and 1,966 in T. Under the predefined differential-expression threshold, the N vs. D comparison yielded more differentially expressed proteins than the D vs. T comparison, suggesting that threshold-detectable proteomic alterations may already be present at dysplasia. Functionally, the N-to-D transition was associated with cell-cycle activation, proliferative signaling, hypoxia-associated responses, and metabolic alterations, whereas the D-to-T transition was more closely related to cytoskeletal organization, motor activity, and structural remodeling. Across the overall N-to-T contrast, glycolysis, pyruvate metabolism, and broader metabolic reprogramming predominated. Trajectory analysis resolved Early Warning, Progression, and dysplasia-restricted transient modules, suggesting a non-linear architecture of proteomic evolution during OSCC progression. MYO1B showed progression-associated spatial increase from N to D and T regions, whereas HSP90AB1 was prioritized as a candidate invasion-associated hub supported by proteomic, protein-protein interaction network, and external transcriptomic analyses. CONCLUSIONS Spatially resolved proteomics of archived formalin-fixed paraffin-embedded tissues provides a morphology-anchored proteomic continuum of oral malignant transformation in a small spatially matched cohort. The data suggest that OSCC progression involves early-shift, progressive, and transient dysplasia-specific programs. These findings offer candidate proteins and biological hypotheses for further validation in larger oral cancer cohorts.

P1.003. Causal Immune-Inflammation Mapping of the GERD-Barrett-Adenocarcinoma Cascade Identifies FGF19-HLA-DR+ T-Cell Axis Driving Esophagogastric Junction Metaplasia

Benign Disease: Barrett’s Esophagus: Pathology and Pathophysiology; Non-invasive Neoplasia Gastroesophageal reflux disease (GERD), Barrett’s esophagus (BE), and esophageal adenocarcinoma (EAC) form a recognized pathological continuum, but the causal immune and inflammatory processes driving progression remain incompletely defined. Summary-level GWAS data were analyzed for 91 circulating inflammatory proteins (n = 14,824), 731 immune cell phenotypes (n = 3,757), and three esophageal diseases (GERD, BE, and EAC). Bidirectional and two-step Mendelian randomization (MR) were used to infer causal effects and mediation, with Cochran’s Q, MR-Egger, and MR-PRESSO applied to assess heterogeneity and pleiotropy. Causal interaction networks were reconstructed to map immune- and inflammation-dominant regulatory patterns across disease stages, and MR-prioritized signals were experimentally validated in EGJ organoids and mouse models. MR supported a causal GERD-BE-EAC sequence, with BE mediating 31.95% of the total GERD-to-EAC effect. In total, 139 immune cell traits and 29 inflammatory proteins showed causal links to disease risk. Mediation analyses highlighted M-CSF1 and HLA-DR+CD4+ T cells as central hubs. Guided by the MR-prioritized FGF19-HLA-DR+ T-cell axis, experimental studies demonstrated that FGF19 promotes esophagogastric junction (EGJ) glandular conversion and increases infiltration of HLA-DR+ CD4+/CD8+ T cells, validated in EGJ organoids and mouse models. This integrated genetic and experimental framework delineates a bidirectional immune-inflammation regulatory network underlying progression from GERD to BE and EAC. FGF19 emerges as a key cytokine driving EGJ glandular remodeling through HLA-DR+ CD4+/CD8+ T-cell associated immune activation, providing candidate molecular targets for early prevention and intervention.

Abstract B039: Genomic and epigenomic complexity underlies barrett’s esophagus progression to adenocarcinoma

Esophageal adenocarcinoma (EAC) is a highly lethal malignancy with rising incidence and poor survival, and is increasingly recognized as a rare but aggressive cancer subtype with limited therapeutic options. Barrett’s esophagus (BE), the only known precursor, progresses through low-grade dysplasia (LGD) and high-grade dysplasia (HGD) to invasive cancer; however, the molecular mechanisms driving this transition remain poorly understood. While genomic alterations have been cataloged, they do not fully explain the dynamic and heterogeneous progression observed in patients. Emerging evidence suggests that epigenetic plasticity is a central driver of this process, particularly in rare cancers where non-genetic mechanisms contribute disproportionately to disease evolution. In this study, we define the role of epigenetic reprogramming in BE-to-EAC progression by integrating spatial transcriptomics, single-cell analyses, and functional modeling. Using high-resolution CosMx spatial molecular imaging, we profiled BE, LGD, HGD, and EAC tissues to map spatially resolved epithelial and microenvironmental cell states. Our data reveal a progressive increase in transcriptional heterogeneity and enhancer-associated gene expression programs across disease stages, accompanied by early DNA methylation changes and widespread chromatin remodeling. Spatial analyses identify distinct epithelial niches characterized by activation of oncogenic signaling pathways, including receptor tyrosine kinase and MYC-driven programs, as well as secretory and inflammatory phenotypes. These epigenetically defined tumor states are tightly coupled to specific stromal and immune microenvironments, suggesting that niche interactions reinforce and stabilize malignant cell states. Integration of single-cell and spatial datasets demonstrates that enhancer activation correlates with transitions from stable epithelial identity to highly plastic, dysplastic, and malignant phenotypes. Functional studies in organoid and in vitro models further support a model in which chronic injury and inflammation drive epigenetic remodeling, leading to enhancer reprogramming and sustained oncogenic transcriptional activation. This epigenetically driven plasticity promotes tumor evolution, cellular heterogeneity, and progression to invasive cancer. Collectively, our findings position epigenetic plasticity as a fundamental mechanism underlying malignant progression in this rare cancer context. By linking enhancer activation to spatially organized tumor states and microenvironmental interactions, this work provides a conceptual and translational framework for identifying actionable epigenetic vulnerabilities. These insights have the potential to inform early detection strategies and enable precision therapeutic interventions to intercept progression in BE and improve outcomes for patients with EAC. Shilpa S. Dhar, Jaffer S. Ajani. Genomic and epigenomic complexity underlies barrett’s esophagus progression to adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr B039.